3′,4′-Isopropylidene-α-galactosylceramide Enables Multidose Th1 Adjuvanticity Despite Limited Serial Serum IFN-γ Responsiveness

Repeated-dose functionality is a critical but often overlooked requirement for invariant natural killer T (iNKT) cell agonists used as vaccine adjuvants. Here, we investigated whether masking the phytosphingosine 3′,4′-diol of α-galactosylceramide (αGC) could alter iNKT agonist behavior in this context. A panel of 3′,4′-protected α-GalCer analogs was synthesized and evaluated for acute systemic cytokine induction, serial serum responsiveness, and adjuvant activity in a three-dose SARS-CoV-2 RBD-Fc vaccination model. Cyclic analogs retained agonist activity, whereas acyclic diacyl-protected analogs were largely inactive. Notably, 3′,4′-carbonate-αGC ( 16 ) induced the strongest acute and serial serum IFN-γ responses, whereas 3′,4′-isopropylidene-αGC ( 17 ) showed attenuated and delayed cytokine kinetics. Despite limited serial serum IFN-γ responsiveness, 17 enhanced antigen-specific IFN-γ by 17-fold over αGC and 6-fold over Alum while maintaining antibody responses. These findings show that conventional acute or serial systemic readouts may not predict repeated-dose vaccine adjuvanticity.